Aop: 23


Each AOP should be given a descriptive title that takes the form “MIE leading to AO”. For example, “Aromatase inhibition [MIE] leading to reproductive dysfunction [AO]” or “Thyroperoxidase inhibition [MIE] leading to decreased cognitive function [AO]”. In cases where the MIE is unknown or undefined, the earliest known KE in the chain (i.e., furthest upstream) should be used in lieu of the MIE and it should be made clear that the stated event is a KE and not the MIE. More help

Androgen receptor agonism leading to reproductive dysfunction (in repeat-spawning fish)

Short name
A short name should also be provided that succinctly summarises the information from the title. This name should not exceed 90 characters. More help
Androgen receptor agonism leading to reproductive dysfunction

Graphical Representation

A graphical summary of the AOP listing all the KEs in sequence, including the MIE (if known) and AO, and the pair-wise relationships (links or KERs) between those KEs should be provided. This is easily achieved using the standard box and arrow AOP diagram (see this page for example). The graphical summary is prepared and uploaded by the user (templates are available) and is often included as part of the proposal when AOP development projects are submitted to the OECD AOP Development Workplan. The graphical representation or AOP diagram provides a useful and concise overview of the KEs that are included in the AOP, and the sequence in which they are linked together. This can aid both the process of development, as well as review and use of the AOP (for more information please see page 19 of the Users' Handbook).If you already have a graphical representation of your AOP in electronic format, simple save it in a standard image format (e.g. jpeg, png) then click ‘Choose File’ under the “Graphical Representation” heading, which is part of the Summary of the AOP section, to select the file that you have just edited. Files must be in jpeg, jpg, gif, png, or bmp format. Click ‘Upload’ to upload the file. You should see the AOP page with the image displayed under the “Graphical Representation” heading. To remove a graphical representation file, click 'Remove' and then click 'OK.'  Your graphic should no longer be displayed on the AOP page. If you do not have a graphical representation of your AOP in electronic format, a template is available to assist you.  Under “Summary of the AOP”, under the “Graphical Representation” heading click on the link “Click to download template for graphical representation.” A Powerpoint template file should download via the default download mechanism for your browser. Click to open this file; it contains a Powerpoint template for an AOP diagram and instructions for editing and saving the diagram. Be sure to save the diagram as jpeg, jpg, gif, png, or bmp format. Once the diagram is edited to its final state, upload the image file as described above. More help


List the name and affiliation information of the individual(s)/organisation(s) that created/developed the AOP. In the context of the OECD AOP Development Workplan, this would typically be the individuals and organisation that submitted an AOP development proposal to the EAGMST. Significant contributors to the AOP should also be listed. A corresponding author with contact information may be provided here. This author does not need an account on the AOP-KB and can be distinct from the point of contact below. The list of authors will be included in any snapshot made from an AOP. More help

Dan Villeneuve, US EPA Mid-Continent Ecology Division (

Point of Contact

Indicate the point of contact for the AOP-KB entry itself. This person is responsible for managing the AOP entry in the AOP-KB and controls write access to the page by defining the contributors as described below. Clicking on the name will allow any wiki user to correspond with the point of contact via the email address associated with their user profile in the AOP-KB. This person can be the same as the corresponding author listed in the authors section but isn’t required to be. In cases where the individuals are different, the corresponding author would be the appropriate person to contact for scientific issues whereas the point of contact would be the appropriate person to contact about technical issues with the AOP-KB entry itself. Corresponding authors and the point of contact are encouraged to monitor comments on their AOPs and develop or coordinate responses as appropriate.  More help
Dan Villeneuve   (email point of contact)


List user names of all  authors contributing to or revising pages in the AOP-KB that are linked to the AOP description. This information is mainly used to control write access to the AOP page and is controlled by the Point of Contact.  More help
  • Dan Villeneuve


The status section is used to provide AOP-KB users with information concerning how actively the AOP page is being developed, what type of use or input the authors feel comfortable with given the current level of development, and whether it is part of the OECD AOP Development Workplan and has been reviewed and/or endorsed. “Author Status” is an author defined field that is designated by selecting one of several options from a drop-down menu (Table 3). The “Author Status” field should be changed by the point of contact, as appropriate, as AOP development proceeds. See page 22 of the User Handbook for definitions of selection options. More help
Author status OECD status OECD project SAAOP status
Open for citation & comment TFHA/WNT Endorsed 1.12 Included in OECD Work Plan
This AOP was last modified on May 13, 2019 10:47
The date the AOP was last modified is automatically tracked by the AOP-KB. The date modified field can be used to evaluate how actively the page is under development and how recently the version within the AOP-Wiki has been updated compared to any snapshots that were generated. More help

Revision dates for related pages

Page Revision Date/Time
Decrease, Population trajectory September 26, 2017 11:33
Agonism, Androgen receptor March 20, 2017 17:44
Reduction, Testosterone synthesis by ovarian theca cells September 16, 2017 10:14
Reduction, 17beta-estradiol synthesis by ovarian granulosa cells September 16, 2017 10:14
Reduction, Plasma 17beta-estradiol concentrations September 26, 2017 11:30
Reduction, Vitellogenin synthesis in liver September 16, 2017 10:16
Reduction, Cumulative fecundity and spawning March 20, 2017 17:52
Reduction, Plasma vitellogenin concentrations September 16, 2017 10:14
Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development September 16, 2017 10:14
Reduction, Gonadotropins, circulating concentrations May 09, 2018 14:05
Agonism, Androgen receptor leads to Reduction, Gonadotropins, circulating concentrations March 20, 2017 11:15
Reduction, Gonadotropins, circulating concentrations leads to Reduction, Testosterone synthesis by ovarian theca cells March 20, 2017 11:24
Reduction, Testosterone synthesis by ovarian theca cells leads to Reduction, 17beta-estradiol synthesis by ovarian granulosa cells March 20, 2017 11:37
Reduction, 17beta-estradiol synthesis by ovarian granulosa cells leads to Reduction, Plasma 17beta-estradiol concentrations March 20, 2017 12:05
Reduction, Plasma 17beta-estradiol concentrations leads to Reduction, Vitellogenin synthesis in liver March 20, 2017 12:28
Reduction, Vitellogenin synthesis in liver leads to Reduction, Plasma vitellogenin concentrations March 20, 2017 12:58
Reduction, Plasma vitellogenin concentrations leads to Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development March 20, 2017 13:21
Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development leads to Reduction, Cumulative fecundity and spawning March 20, 2017 13:35
Reduction, Cumulative fecundity and spawning leads to Decrease, Population trajectory March 20, 2017 13:49
Agonism, Androgen receptor leads to Reduction, Testosterone synthesis by ovarian theca cells March 20, 2017 15:55
Agonism, Androgen receptor leads to Reduction, 17beta-estradiol synthesis by ovarian granulosa cells March 20, 2017 15:56
Agonism, Androgen receptor leads to Reduction, Vitellogenin synthesis in liver March 20, 2017 15:59
Reduction, Plasma 17beta-estradiol concentrations leads to Reduction, Plasma vitellogenin concentrations October 18, 2018 11:02
17beta-Trenbolone November 29, 2016 18:42


In the abstract section, authors should provide a concise and informative summation of the AOP under development that can stand-alone from the AOP page. Abstracts should typically be 200-400 words in length (similar to an abstract for a journal article). Suggested content for the abstract includes the following: The background/purpose for initiation of the AOP’s development (if there was a specific intent) A brief description of the MIE, AO, and/or major KEs that define the pathway A short summation of the overall WoE supporting the AOP and identification of major knowledge gaps (if any) If a brief statement about how the AOP may be applied (optional). The aim is to capture the highlights of the AOP and its potential scientific and regulatory relevance More help

This adverse outcome pathway details the linkage between binding and activation of androgen receptor as a nuclear transcription factor in females and reproductive dysfunction as evidenced through reductions cumulative fecundity and spawning in repeat-spawning fish species.  Androgen receptor mediated activities are one of the major activities of concern to endocrine disruptor screening programs worldwide.  Cumulative fecundity is the most apical endpoint considered in the OECD 229 Fish Short Term Reproduction Assay. The OECD 229 assay serves as screening assay for endocrine disruption and associated reproductive impairment (OECD 2012). Cumulative fecundity is one of several variables known to be of demographic significance in forecasting fish population trends. Therefore, this AOP has utility in supporting the application of measures of androgen receptor binding and activation as a nuclear transcription factor as a means to identify chemicals with known potential to adversely affect fish populations. At present this AOP is largely supported by evidence conducted with small laboratory model fish species such as Pimephales promelas, Oryzias latipes, and Fundulus heteroclitus. While many aspects of the biology underlying this AOP are largely conserved across vertebrates, particularly oviparous vertebrates, the relevance of this AOP to vertebrate classes other than fish as well as to fish species employing different reproductive strategies has not been established at this time. Thus, caution should be used in applying this AOP beyond a fairly narrow range of fish species with life cycles similar to that of the three species noted above.

Background (optional)

This optional subsection should be used to provide background information for AOP reviewers and users that is considered helpful in understanding the biology underlying the AOP and the motivation for its development. The background should NOT provide an overview of the AOP, its KEs or KERs, which are captured in more detail below. Examples of potential uses of the optional background section are listed on pages 24-25 of the User Handbook. More help

No additional background

Summary of the AOP

This section is for information that describes the overall AOP. The information described in section 1 is entered on the upper portion of an AOP page within the AOP-Wiki. This is where some background information may be provided, the structure of the AOP is described, and the KEs and KERs are listed. More help


Molecular Initiating Events (MIE)
An MIE is a specialised KE that represents the beginning (point of interaction between a stressor and the biological system) of an AOP. More help
Key Events (KE)
This table summarises all of the KEs of the AOP. This table is populated in the AOP-Wiki as KEs are added to the AOP. Each table entry acts as a link to the individual KE description page.  More help
Adverse Outcomes (AO)
An AO is a specialised KE that represents the end (an adverse outcome of regulatory significance) of an AOP.  More help
Sequence Type Event ID Title Short name
1 MIE 25 Agonism, Androgen receptor Agonism, Androgen receptor
2 KE 129 Reduction, Gonadotropins, circulating concentrations Reduction, Gonadotropins, circulating concentrations
3 KE 274 Reduction, Testosterone synthesis by ovarian theca cells Reduction, Testosterone synthesis by ovarian theca cells
4 KE 3 Reduction, 17beta-estradiol synthesis by ovarian granulosa cells Reduction, 17beta-estradiol synthesis by ovarian granulosa cells
5 KE 219 Reduction, Plasma 17beta-estradiol concentrations Reduction, Plasma 17beta-estradiol concentrations
6 KE 285 Reduction, Vitellogenin synthesis in liver Reduction, Vitellogenin synthesis in liver
7 KE 221 Reduction, Plasma vitellogenin concentrations Reduction, Plasma vitellogenin concentrations
8 KE 309 Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development Reduction, Vitellogenin accumulation into oocytes and oocyte growth/development
9 KE 78 Reduction, Cumulative fecundity and spawning Reduction, Cumulative fecundity and spawning
10 AO 360 Decrease, Population trajectory Decrease, Population trajectory

Relationships Between Two Key Events (Including MIEs and AOs)

This table summarises all of the KERs of the AOP and is populated in the AOP-Wiki as KERs are added to the AOP. Each table entry acts as a link to the individual KER description page.To add a key event relationship click on either Add relationship: events adjacent in sequence or Add relationship: events non-adjacent in sequence.For example, if the intended sequence of KEs for the AOP is [KE1 > KE2 > KE3 > KE4]; relationships between KE1 and KE2; KE2 and KE3; and KE3 and KE4 would be defined using the add relationship: events adjacent in sequence button.  Relationships between KE1 and KE3; KE2 and KE4; or KE1 and KE4, for example, should be created using the add relationship: events non-adjacent button. This helps to both organize the table with regard to which KERs define the main sequence of KEs and those that provide additional supporting evidence and aids computational analysis of AOP networks, where non-adjacent KERs can result in artifacts (see Villeneuve et al. 2018; DOI: 10.1002/etc.4124).After clicking either option, the user will be brought to a new page entitled ‘Add Relationship to AOP.’ To create a new relationship, select an upstream event and a downstream event from the drop down menus. The KER will automatically be designated as either adjacent or non-adjacent depending on the button selected. The fields “Evidence” and “Quantitative understanding” can be selected from the drop-down options at the time of creation of the relationship, or can be added later. See the Users Handbook, page 52 (Assess Evidence Supporting All KERs for guiding questions, etc.).  Click ‘Create [adjacent/non-adjacent] relationship.’  The new relationship should be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. To edit a key event relationship, click ‘Edit’ next to the name of the relationship you wish to edit. The user will be directed to an Editing Relationship page where they can edit the Evidence, and Quantitative Understanding fields using the drop down menus. Once finished editing, click ‘Update [adjacent/non-adjacent] relationship’ to update these fields and return to the AOP page.To remove a key event relationship to an AOP page, under Summary of the AOP, next to “Relationships Between Two Key Events (Including MIEs and AOs)” click ‘Remove’ The relationship should no longer be listed on the AOP page under the heading “Relationships Between Two Key Events (Including MIEs and AOs)”. More help
Title Adjacency Evidence Quantitative Understanding

Network View

The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help


The stressor field is a structured data field that can be used to annotate an AOP with standardised terms identifying stressors known to trigger the MIE/AOP. Most often these are chemical names selected from established chemical ontologies. However, depending on the information available, this could also refer to chemical categories (i.e., groups of chemicals with defined structural features known to trigger the MIE). It can also include non-chemical stressors such as genetic or environmental factors. Although AOPs themselves are not chemical or stressor-specific, linking to stressor terms known to be relevant to different AOPs can aid users in searching for AOPs that may be relevant to a given stressor. More help
Name Evidence Term
17beta-Trenbolone High

Life Stage Applicability

Identify the life stage for which the KE is known to be applicable. More help
Life stage Evidence
Adult, reproductively mature

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
Pimephales promelas Pimephales promelas NCBI

Sex Applicability

The authors must select from one of the following: Male, female, mixed, asexual, third gender, hermaphrodite, or unspecific. More help
Sex Evidence

Overall Assessment of the AOP

This section addresses the relevant biological domain of applicability (i.e., in terms of taxa, sex, life stage, etc.) and WoE for the overall AOP as a basis to consider appropriate regulatory application (e.g., priority setting, testing strategies or risk assessment). The goal of the overall assessment is to provide a high level synthesis and overview of the relative confidence in the AOP and where the significant gaps or weaknesses are (if they exist). Users or readers can drill down into the finer details captured in the KE and KER descriptions, and/or associated summary tables, as appropriate to their needs.Assessment of the AOP is organised into a number of steps. Guidance on pages 59-62 of the User Handbook is available to facilitate assignment of categories of high, moderate, or low confidence for each consideration. While it is not necessary to repeat lengthy text that appears elsewhere in the AOP description (or related KE and KER descriptions), a brief explanation or rationale for the selection of high, moderate, or low confidence should be made. More help
Attached file: Annex1 for aop 23 ar reproductive dys 2017 03 20

Annex 1 Table, Assessment of the relative level of confidence in the overall AOP based on rank ordered weight of evidence elements is attached in PDF format.

Domain of Applicability

The relevant biological domain(s) of applicability in terms of sex, life-stage, taxa, and other aspects of biological context are defined in this section. Biological domain of applicability is informed by the “Description” and “Biological Domain of Applicability” sections of each KE and KER description (see sections 2G and 3E for details). In essence the taxa/life-stage/sex applicability is defined based on the groups of organisms for which the measurements represented by the KEs can feasibly be measured and the functional and regulatory relationships represented by the KERs are operative.The relevant biological domain of applicability of the AOP as a whole will nearly always be defined based on the most narrowly restricted of its KEs and KERs. For example, if most of the KEs apply to either sex, but one is relevant to females only, the biological domain of applicability of the AOP as a whole would be limited to females. While much of the detail defining the domain of applicability may be found in the individual KE and KER descriptions, the rationale for defining the relevant biological domain of applicability of the overall AOP should be briefly summarised on the AOP page. More help

Domain(s) of Applicability

Chemical: This AOP applies to non-aromatizable androgens. Compounds which can bind the AR in vitro, but are converted to high potency estrogens in vivo through aromatization do not produce the profile of effects described in the present AOP (e.g., methyltestosterone [Ankley et al. 2001; Pawlowski et al. 2004]; androstenedione [OECD 2007]).

Sex: The AOP applies to females only.

Life stages: The relevant life stages for this AOP are reproductively mature adults. This AOP does not apply to adult stages that lack a sexually mature ovary, for example as a result of seasonal or environmentally-induced gonadal senescence (i.e., through control of temperature, photo-period, etc. in a laboratory setting).

Taxonomic: At present, the assumed taxonomic applicability domain of this AOP is iteroparous teleost fish species.

  • However, to date the majority of toxicological data on which this AOP is based has been limited to several small fish species, fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), and mummichog (Fundulus heteroclitus) with asynchronous oocyte development and a repeat spawning reproductive strategy.
  • Species dependent differences in endocrine feedback responses, likely associated with different reproductive strategies, have been reported. Thus, the applicability domain may prove more restricted than currently assumed. In particular, the applicability to fish species with synchronous or group synchronous oocyte development patterns (see Wallace and Selman 1981) is unclear.
  • European eel may be an exception to the generalizability of the negative feedback response to a non-aromatizable xenoandrogen (Huang et al. 1997).
  • Reductions in plasma VTG concentrations and/or hepatic VTG mRNA abundance in females following exposure to 17β-trenbolone has been observed in Pimephales promelas, Oryzias latipes, Danio rerio, (Seki et al. 2006), Cyprinodon variegatus (Hemmer et al. 2008), Gambusia holbrooki and Gambusia affinis (Brockmeier et al. 2013)

[Assessment provided by Ioanna Katsiadaki - reviewer]:  This is restricted clearly to female fish only as adversity is linked to reduced oestrogen synthesis (via reduced androgen synthesis); it is also limited to fully reproductive mature fish (not fish entering puberty or juvenile fish) and importantly is limited to fish that once they reach sexual maturity they spawn constantly. The latter is a reproductive strategy employed by fish that tend to occupy tropical areas (around the equator). Unfortunately most fish species have different reproductive strategies (annual life cycle) hence the level of gonadotropin expression (and consequently steroid production) is regulated by photoperiodic and temperature changes throughout the year. Even if a negative feedback mechanism operates in all of these species and in all life stages (which is certainly not the case) we still need to establish what is the relative strength of the AR agonist induced negative feedback to the environment-induced stimulation of gonadotropins! This link has never been studied and is critical if we really mean to protect wildlife.

Essentiality of the Key Events

An important aspect of assessing an AOP is evaluating the essentiality of its KEs. The essentiality of KEs can only be assessed relative to the impact of manipulation of a given KE (e.g., experimentally blocking or exacerbating the event) on the downstream sequence of KEs defined for the AOP. Consequently evidence supporting essentiality is assembled on the AOP page, rather than on the independent KE pages that are meant to stand-alone as modular units without reference to other KEs in the sequence.The nature of experimental evidence that is relevant to assessing essentiality relates to the impact on downstream KEs and the AO if upstream KEs are prevented or modified. This includes: Direct evidence: directly measured experimental support that blocking or preventing a KE prevents or impacts downstream KEs in the pathway in the expected fashion. Indirect evidence: evidence that modulation or attenuation in the magnitude of impact on a specific KE (increased effect or decreased effect) is associated with corresponding changes (increases or decreases) in the magnitude or frequency of one or more downstream KEs.When assembling the support for essentiality of the KEs, authors should organise relevant data in a tabular format. The objective is to summarise briefly the nature and numbers of investigations in which the essentiality of KEs has been experimentally explored either directly or indirectly. See pages 50-51 in the User Handbook for further definitions and clarifications.  More help
  • In general, few studies have directly addressed the essentiality of the proposed sequence of key events.
  • Ekman et al. 2011 provide evidence that in fathead minnow, cessation of trenbolone exposure resulted in recovery of plasma E2 and VTG concentrations which were depressed by continuous exposure to 17beta trenbolone. This provides some support for the essentiality of these two key events.
  • Essentiality of the proposed negative feedback key event is supported by experimental work that evaluated the ability of AR agonists to reduce T or E2 production in vitro. There are no known reports of 17β-trenbolone directly inhibiting steroid biosynthesis. When tested in an in vitro steroidogenesis assay using H295R adrenal carcinoma cells, trenbolone caused a concentration-dependent increase in estradiol production, as opposed to any reductions in steroid hormone concentrations, an effect that was concurrent with increased transcription of CYP19 (aromatase) in the cell line (Gracia et al. 2007).

Evidence Assessment

The biological plausibility, empirical support, and quantitative understanding from each KER in an AOP are assessed together.  Biological plausibility of each of the KERs in the AOP is the most influential consideration in assessing WoE or degree of confidence in an overall hypothesised AOP for potential regulatory application (Meek et al., 2014; 2014a). Empirical support entails consideration of experimental data in terms of the associations between KEs – namely dose-response concordance and temporal relationships between and across multiple KEs. It is examined most often in studies of dose-response/incidence and temporal relationships for stressors that impact the pathway. While less influential than biological plausibility of the KERs and essentiality of the KEs, empirical support can increase confidence in the relationships included in an AOP. For clarification on how to rate the given empirical support for a KER, as well as examples, see pages 53- 55 of the User Handbook.  More help

Biological Plausibility

  • The biochemistry of steroidogenesis and the predominant role of the gonad in synthesis of the sex steroids are well established.
  • Similarly, the role of E2 as the major regulator of hepatic vitellogenin production is widely documented in the literature.
  • The direct link between reduced VTG concentrations in the plasma and reduced uptake into oocytes is highly plausible, as the plasma is the primary source of the VTG.
  • The direct connection between reduced VTG uptake and impaired spawning/reduced cumulative fecundity is more tentative. It is not clear, for instance whether impaired VTG uptake limits oocyte growth and failure to reach a critical size in turn impairs physical or inter-cellular signaling processes that promote release of the oocyte from the surrounding follicles. In at least one experiment, oocytes with similar size to vitellogenic oocytes, but lacking histological staining characteristic of vitellogenic oocytes was observed (R. Johnson, personal communication). At present, the link between reductions in circulating VTG concentrations and reduced cumulative fecundity are best supported by the correlation between those endpoints across multiple experiments, including those that impact VTG via other molecular initiating events (Miller et al. 2007).
  • At present, negative feedback is the most biologically plausible explanation for the reductions in ex vivo T and E2 production following exposure to 17β-trenbolone.There are no known reports of 17β-trenbolone directly inhibiting steroid biosynthesis. When tested in an in vitro steroidogenesis assay using H295R adrenal carcinoma cells, trenbolone caused a concentration-dependent increase in estradiol production, as opposed to any reductions in steroid hormone concentrations, an effect that was concurrent with increased transcription of CYP19 (aromatase) in the cell line (Gracia et al. 2007). Given the lack of any established direct effect on steroidogenic enzyme activity, negative feedback is currently the most likely explanation for the consistent effects observed in vivo. That said, many uncertainties regarding the exact mechanisms through which an exogenous, non-aromatizable, AR agonist elicits negative feedback remain.

Concordance of dose-response relationships:  See Concordance Table  (available in Excel and PDF format)

There are a limited number of studies in which multiple key events were considered in the same study following exposure to known, non-aromatizable, AR agonists. These were considered the most useful for evaluating the concordance of dose-response relationships. In general, effects on downstream key events occurred at concentrations equal to or greater than those at which upstream events occurred. For exposures to 17b-trenbolone, key events related to steroid production and circulating estradiol and vitellogenin concentrations were impacted at the same dose at which effects on cumulative fecundity were observed. Effects on vitellogenin transcription were only observed at greater concentrations, but data for comparable species and dose ranges were unavailable at present. For two other AR agonists tested in fish, available studies examined a single time-point only. Consequently, it was unclear whether lower effect concentrations for certain downstream KEs, relative to upstream were due to a lack of dose-response concordance, or due to decreased sensitivity of the upstream later in the exposure time-course.

While not directly addressing dose-response concordance, the dependence of the key events on the concentration of the androgen agonist has been established for all key events starting at and down-stream of reduced T synthesis. However, to date we are not aware of any studies that have established a concentration-response relationship between exposure to non-endogenous AR agonists (e.g., xenobiotics, pharmaceuticals) and circulating gonadotropin concentrations in fish or other vertebrates.

  • Exposure of female fathead minnows to the AR agonist 17β-trenbolone for 21 d caused concentration-dependent reductions in circulating T, E2, and VTG concentrations over a range from 0.005 to 0.5 μg/L. The concentration response for all three variables had a “U”-shaped concentration response curve which may indicate concentration-dependent differences in the feedback response and/or compensatory processes. Histological evidence of reduced VTG uptake and reduced gonad stage were evident, although the concentration-response of histological effects was not determined. Despite the “U”-shaped concentration-response at the biochemical level, concentration-dependent reductions in cumulative fecundity were observed (Ankley et al. 2003). Effective concentrations were consistent with those causing phenotypic masculinization in female fish.
  • Jensen et al. (2006) also demonstrated concentration-dependent reductions in circulating T, E2, and VTG following 21 d of in vivo exposure to 17α-trenbolone (Jensen et al. 2006).
  • In a time-course experiment in which female fathead minnows were exposed to to 33 or 472 ng 17β-trenbolone/L ex vivo T, ex vivo E2, plasma E2, and plasma VTG all showed concentration-dependent reductions that were consistent with the AOP (Ekman et al. 2011).
  • Exposure of female fathead minnows to spironolactone, a pharmaceutical that binds the fathead minnow AR, for 21 d caused concentration-dependent reductions in cumulative fecundity, plasma VTG and VTG mRNA expression, and plasma E2 concentrations. The frequency and severity of females with decreased yolk accumulation, and increased oocyte atresia was concentration-dependent. The chemical also induced phenotypic masculinization in female fish. (Lalone et al. 2013).
  • Exposure of female medaka to spironolactone caused concentration-dependent reductions in cumulative fecundity and VTG mRNA expression (impacts on steroid hormone concentrations were not measured). Spironolactone also caused phenotypic masculinization of female medaka (Lalone et al. 2013).

Temporal concordance among the key events and adverse effect: Temporal concordance between activation of the AR as a nuclear transcription factor and onset of a negative feedback response resulting in decreased gonadotropin secretion has not been established. Temporal concordance of the key events starting with reduced T biosynthesis and proceeding through reductions in plasma vitellogenin has been established (Concordance Table). Temporal concordance beyond the key event of reductions in plasma vitellogenin has not been established, in large part due to disconnect in the time-scales over which the events can be measured. For example, most small fish used in reproductive toxicity testing can spawn anywhere from once daily to several days per week. Given the variability in daily spawning rates, it is neither practical nor effective to evaluate cumulative fecundity at a time scale shorter than roughly a week. Since the impacts at lower levels of biological organization can be detected within hours of exposure, lack of impact on cumulative fecundity before the other key events are impacted cannot be effectively measured. Overall, among those key events whose temporal concordance can reasonably be evaluated based on currently available data, the temporal profile observed is consistent with the AOP.

Consistency: We are aware of no cases where the pattern of key events described was observed without also observing a significant impact on cumulative fecundity. Due to variability in the cumulative fecundity endpoint and potential compensatory responses ((Villeneuve et al. 2009; Villeneuve et al. 2013; Ankley et al. 2009b; Zhang et al. 2008; Ekman et al. 2012), the cumulative fecundity endpoint can be less sensitive than key events measured at lower levels of biological organization. Nonetheless, the occurrence of the final adverse outcome when the other key events are observed is very consistent. The final adverse effect is not specific to this AOP. Many of the key events included in this AOP overlap with AOPs linking other molecular initiating events to reproductive dysfunction in small fish.

  • In general, there is a consistent body of evidence linking exposure to an AR agonist to decreased T synthesis, E2 synthesis, circulating E2 and VTG concentrations, and cumulative fecundity in female fish. For example, the association between 17β-trenbolone exposure and reduced vitellogenin concentrations in females has been replicated in over a dozen independent experiments (Ekman et al. 2011; Ankley et al. 2003; Jensen et al. 2006; Ankley et al. 2010; Hemmer et al. 2008; Seki et al. 2006; Brockmeier et al. 2013). However, relatively few exogenous, non-aromatizable, AR agonists have been tested. Other than recent work with spironolactone (Lalone et al. 2013), we are not aware of the profile of responses being demonstrated for other AR agonists.

Uncertainties, inconsistencies, and data gaps: There are three major areas of uncertainty and data gaps in the current AOP: 

  • First, there remains considerable uncertainty as to the specific mechanism(s) through which AR agonism elicits a negative feedback response at the level of the hypothalamus and/or pituitary. There is also a substantial data gap relative to establishing that exposure to an AR agonist like 17β-trenbolone causes concentration-dependent reductions in circulating gonadotropins. That uncertainty is amplified further by the variation in feedback control along the endocrine axis for fish species employing different reproductive strategies. For example, gonadotropin regulation may be very different in species with synchronous oocyte maturation and annual or once per life-time reproductive strategies. Thus, there are considerable uncertainties related to the taxonomic relevance of this AOP to a broader range of fish species or other vertebrates.
  • The second major uncertainty in this AOP relates to whether there is a direct biological linkage between impaired VTG uptake into oocytes and impaired spawning/reduced cumulative fecundity. Plausible biological connections have been hypothesized, but have not yet been tested experimentally.
  • A third uncertainty pertains to the chemical domain of applicability. In vivo, a number of chemicals that are detected as androgens in in vitro screening assays such as receptor binding assays or ligand-activated transcriptional assay can be aromatized to functional estrogens. Thus, in vivo such compounds may produce a profile of effects more consistent with estrogen receptor activation than AR activation or may produced mixed effects characteristic of either estrogen or androgen exposures (e.g., Pawlowski et al. 2004; Hornung et al. 2004). Examples of such aromatizable androgens include, testosterone, methyltestosterone, and androstenedione. Consequently, caution is warranted in applying this AOP based on in vitro screening data alone, without consideration for possible conversion to estrogens.

Quantitative Understanding

Some proof of concept examples to address the WoE considerations for AOPs quantitatively have recently been developed, based on the rank ordering of the relevant Bradford Hill considerations (i.e., biological plausibility, essentiality and empirical support) (Becker et al., 2017; Becker et al, 2015; Collier et al., 2016). Suggested quantitation of the various elements is expert derived, without collective consideration currently of appropriate reporting templates or formal expert engagement. Though not essential, developers may wish to assign comparative quantitative values to the extent of the supporting data based on the three critical Bradford Hill considerations for AOPs, as a basis to contribute to collective experience.Specific attention is also given to how precisely and accurately one can potentially predict an impact on KEdownstream based on some measurement of KEupstream. This is captured in the form of quantitative understanding calls for each KER. See pages 55-56 of the User Handbook for a review of quantitative understanding for KER's. More help

Assessment of quantitative understanding of the AOP: At present, the quantitative understanding of the AOP is insufficient to directly link a measure of chemical potency as an AR agonist (e.g., as measured in a transcriptional activation assay) to a predicted effect concentration at the level of cumulative fecundity. However, a number of mechanistic and statistical models are sufficiently developed to facilitate predictions of cumulative outcomes based on intermediate key event measurements such as circulating vitellogenin concentrations. Because the current models were developed based on a fairly limited range of model compounds and species, the general applicability and degree of accuracy and precision in the model-derived predictions remains uncertain.

Considerations for Potential Applications of the AOP (optional)

At their discretion, the developer may include in this section discussion of the potential applications of an AOP to support regulatory decision-making. This may include, for example, possible utility for test guideline development or refinement, development of integrated testing and assessment approaches, development of (Q)SARs / or chemical profilers to facilitate the grouping of chemicals for subsequent read-across, screening level hazard assessments or even risk assessment. While it is challenging to foresee all potential regulatory application of AOPs and any application will ultimately lie within the purview of regulatory agencies, potential applications may be apparent as the AOP is being developed, particularly if it was initiated with a particular application in mind. This optional section is intended to provide the developer with an opportunity to suggest potential regulatory applications and describe his or her rationale.To edit the “Considerations for Potential Applications of the AOP” section, on an AOP page, in the upper right hand menu, click ‘Edit.’ This brings you to a page entitled, “Editing AOP.” Scroll down to the “Considerations for Potential Applications of the AOP” section, where a text entry box allows you to submit text. In the upper right hand menu, click ‘Update AOP’ to save your changes and return to the AOP page or 'Update and continue' to continue editing AOP text sections.  The new text should appear under the “Considerations for Potential Applications of the AOP” section on the AOP page. More help



List the bibliographic references to original papers, books or other documents used to support the AOP. More help
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  • Ankley GT, Cavallin JE, Durhan EJ, Jensen KM, Kahl MD, Makynen EA, et al. 2012. A time-course analysis of effects of the steroidogenesis inhibitor ketoconazole on components of the hypothalamic-pituitary-gonadal axis of fathead minnows. Aquatic toxicology 114-115: 88-95.
  • Ankley GT, Jensen KM, Durhan EJ, Makynen EA, Butterworth BC, Kahl MD, et al. 2005. Effects of two fungicides with multiple modes of action on reproductive endocrine function in the fathead minnow (Pimephales promelas). Toxicol Sci 86(2): 300-308.
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  • Ankley GT, Jensen KM, Kahl MD, Korte JJ, Makynen EA. 2001. Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environ Toxicol Chem 20:1276-1290.
  • Ankley GT, Jensen KM, Kahl MD, Makynen EA, Blake LS, Greene KJ, et al. 2007. Ketoconazole in the fathead minnow (Pimephales promelas): reproductive toxicity and biological compensation. Environ Toxicol Chem 26(6): 1214-1223.
  • Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, et al. 2003. Effects of the androgenic growth promoter 17-b-trenbolone on fecundity and reproductive endocrinology of the fathead minnow. Environmental Toxicology and Chemistry 22(6): 1350-1360.
  • Ankley GT, Kahl MD, Jensen KM, Hornung MW, Korte JJ, Makynen EA, et al. 2002. Evaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas). Toxicological Sciences 67: 121-130.
  • Ankley GT, Miller DH, Jensen KM, Villeneuve DL, Martinovic D. 2008. Relationship of plasma sex steroid concentrations in female fathead minnows to reproductive success and population status. Aquatic toxicology 88(1): 69-74.
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